Open Access
Review
Generating and manipulating transgenic animals using transposable
elements
David A Largaespada*
Address: Department of Genetics, Cell Biology and Development, University of Minnesota Cancer Center, Minneapolis, MN 55455, USA
Email: David A Largaespada* - [email protected] * Corresponding author
Abstract
Transposable elements, or transposons, have played a significant role in the history of biological research. They have had a major influence on the structure of genomes during evolution, they can cause mutations, and their study led to the concept of so-called "selfish DNA". In addition, transposons have been manipulated as useful gene transfer vectors. While primarily restricted to use in invertebrates, prokaryotes, and plants, it is now clear that transposon technology and biology are just as relevant to the study of vertebrate species. Multiple transposons now have been shown to be active in vertebrates and they can be used for germline transgenesis, somatic cell transgenesis/ gene therapy, and random germline insertional mutagenesis. The sophistication of these applications and the number of active elements are likely to increase over the next several years. This review covers the vertebrate-active retrotransposons and transposons that have been well studied and adapted for use as gene transfer agents. General considerations and predictions about the future utility of transposon technology are discussed.
Introduction
"One man's trash is another man's treasure." Anonymous
Many vertebrate genomes, including the human genome, are littered with trash, in the form of so-called "junk DNA". Only a small portion of most vertebrate genomes has an obvious biological role for the organism, such as protein encoding open reading frames, untranslated regions (UTR) of mRNA encoding DNA, promoter sequences, or ribosomal RNA encoding DNA. Instead, the vast majority of vertebrate genomic DNA has no obvious function and is indeed dominated by the presence of repetitive DNA elements that are themselves the remnants of transposable elements or the result of transposable ele-ment activity. Some of these eleele-ments have become inac-tive during the evolution of today's species, some are dependent upon enzymes encoded in trans by other trans-posable elements, and some remain active. These
sequences are interesting for a variety of reasons. They can be used as markers of a species evolutionary history, they can be involved in the creation and destruction of genes, transposase genes have been "adopted" by some organ-isms to perform vital cellular functions (such as V-D-J recombination), and finally, transposons have been used as a source of vectors for transgenesis and mutagenesis in multiple species. It is this last feature of transposable ele-ments, in particular their use in vertebrate species, that is the concern of this review. While the history of transpo-son study and vector development for use in bacteria, fungi, plant, and various invertebrate species is very long, it has been only very recently that transposable elements have demonstrated effectiveness as genetic tools in verte-brate species. We speculate that these elements will become important tools for vertebrate germline
Published: 07 November 2003
Reproductive Biology and Endocrinology 2003, 1:80
Received: 15 July 2003 Accepted: 07 November 2003
This article is available from: http://www.rbej.com/content/1/1/80
transgenesis, somatic and germline mutagenesis, and human gene therapy.
Transposable Elements
Many excellent reviews have been written on the subject of transposable elements and it is not the goal of this
review to systematically cover that very large field, which really would require a textbook to do the subject justice [1–3]. It can be said, however, that transposons come in two general types (Figure 1). The "copy and paste" retro-transposons are mobilized by transcribing an RNA copy, that then becomes reverse transcribed and is integrated elsewhere in the genome. In contrast, the "cut and paste" transposable elements transpose by the direct excision from DNA and insertion elsewhere in the genome. Both types of elements have now been used in vertebrate cell lines and animals as gene transfer vectors. Retrotrans-posons come in many classes and include retroviruses, which will not be considered in this review. Retrotranspo-son elements require at a minimum, an internal promoter and coding sequences for an integrase and reverse tran-scriptase protein. Many of these elements encode proteins that act primarily in cis, on the RNA transcript from which they were translated. Thus, in biotechnology applications, foreign gene sequences must be added to the 3' untrans-lated region (UTR) of the retrotransposon vector (Figure 1). In contrast, the cut and paste transposases can act in trans. These elements have an internal promoter and encode a single protein "transposase" which binds to ter-minal repeat sequences on the ends of the element, caus-ing it to be excised and inserted elsewhere. Because the cut and paste transposons can act in trans, it is feasible to sup-ply the transposase by a variety of methods (DNA, RNA and possibly transferred protein) and insert any desired sequence in the transposon vector DNA itself (Figure 1). These sequences might include genes for germline or somatic cell transgenesis, sequences for insertional muta-genesis, or recognition by site-specific recombinases.
What follows is a review of recent success in developing vertebrate gene transfer vectors based on retrotransposons or DNA transposons. Currently these vector systems are at different levels of development and come from a variety of sources (Table 1). Some are elements taken from inver-tebrate species and adapted for use in verinver-tebrates. Some are endogenous, naturally active vertebrate elements, while one was "reconstructed" from study of a large number of endogenous defective elements. Some of these vector systems have been shown to be effective for germ-line and somatic cell transgenesis in vivo, while others have so far only been shown to be active in cultured ver-tebrate cell lines. Finally, some have been introduced into transgenic mice and been mobilized from chromosomally resident positions. Thus, the transposons may be useful as general germline insertional mutagens. It is clear that mul-tiple transposons, derived from various sources, might find utility for generating and manipulating transgenic animals. Because each vector system has advantages and disadvantages, depending on the specific application, multiple vector systems should be developed for use in the future.
"Copy-and-paste" and "cut-and-paste" transposons have been adapted for use as gene transfer vectors
Figure 1
General Considerations for Transposable Elements in Scientific and Biotechnology Applications
Transposons have the useful property of catalyzing the most important step in gene transfer applications, the insertion of foreign DNA into host chromosomes. A vari-ety of uses for transposons in vertebrate science and bio-technology can be imagined. However, so far three general uses have been explored: germline transgenesis, somatic cell transgenesis/gene therapy, and random inser-tional mutagenesis (Figure 2).
First of all, germline transgenesis by transposition has been developed for certain of the transposon systems that are active in vertebrates. This has involved the co-delivery of in vitro transcribed mRNA encoding the transposase with transposon vector DNA into early embryos of the frog, Xenopus tropicailis, the zebrafish Dana rario, or the mouse (Paul Mead and Steve Ekker, personal communica-tion) [4]. In the case of mariner-mediated transformation of the chicken germline, an active autonomous DNA ele-ment was injected [5]. It remains to be determined if Table 1: Transposable elements active in vertebrate species for use as gene transfer and insertional mutagenesis vectors.
Transposon Familya Activity in cultured cellsb Activity in vertebrate animalsc References
L1 LINE retrotransposon Mouse (PCT) Human (PCT) Mouse (TCRG) [21,22] Tol2 hAT Mouse (EP) Human (EP) Medaka (TCRG) Zebrafish (TCRG). [27,28,32] Tc1 Tc1/mariner Human (PCT) Mouse (PCT) - [19,39,40] Tc3 Tc1/mariner Human (PCT) Zebrafish (GT and TCRG) [19,42] Mariner (Himar1) Tc1/mariner Human (IPT) - [19,44] Mariner (Mos1) Tc1/mariner Human (PCT) Chicken (GT), zebrafish (GT) [5,19,43] Minos Tc1/mariner Human (PCT) Mouse (TCRG) Mouse (TCRS) [46–48] Sleeping Beauty Tc1/mariner Mouse, hamster, human, monkey,
dog, cow, sheep, quail, Xenopus, many fish (PCT)
Mouse (GT) Mouse lung, liver (SCT) Mouse (TCRG) Mouse (TCRS)
[4,19,30,51,53–56,59,60]
aRelevant superfamily of transposable element. bActivity in immortalized cell lines has been demonstrated using by demonstrating excision from an introduced plasmid (EP), interplasmid transposition (IPT), or full transposition from introduced plasmid (or viral vector) into chromosomes (PCT). cActivity in intact animals (various species) has been demonstrated by germline transgenesis (GT), somatic cell transgenesis (SCT), transposition of chromosomally resident transposon vectors in the germline (TCRG), and transposition of chromosomally resident transposon vectors in the soma (TCRS).
General uses for transposon vectors in the generation and manipulation of transgenic animals
Figure 2
transgenesis in commercially important species such as the chicken, pig, goat or cow can be improved using trans-posons, but this area is certainly worth exploring given the success in diverse species so far. Given the complexities of harvesting fertilized embryos from large animals, thought also should be given to sperm transgenesis, since this may offer many practical benefits. Certain advantages can be imagined for achieving germline transgenesis by transpo-sition, as compared to standard pronuclear injection of DNA. One advantage is the ability, in some cases, to gen-erate offspring with multiple independent insertions that can then be segregated by breeding, thus increasing the number of total potentially useful transgenesis events. Standard pronuclear injection of linear DNA into embryos results in concatomerization of the DNA and random integration into the genome. However, these con-catomer integration events are often associated with inver-sions, deletions or other large rearrangements of DNA at the integration site. Indeed, for this reason, up to 10% of all mouse germline transgenics have a distinct phenotype in the homozygous state due to insertional mutation/ deletion of endogenous genes [6]. Although these events have allowed the identification of important endogenous genes, it is in general an undesired effect of germline trans-genesis. The ability of transposons to deliver a specific fragment of DNA into a target site without alteration of endogenous sequences, other than the insertion of the transposon vector, could thus be considered an advan-tage. One disadvantage of transgenesis by transposition, could be the fact that multiple copies of a transgene can-not be integrated into one position by transgenesis, and so it may be difficult in some cases to achieve very high expression of transgenes by this method. Very low expres-sion is a frequent problem for any transgenic project. Since transgene arrays are often subject to partial methyl-ation and inactivmethyl-ation, as well as other mechanisms of silencing, often only a multicopy array can achieve expres-sion levels rivaling the endogenous gene. That is, small transgene vectors typically express at a level that is only a fraction of the level of the native gene. Nevertheless, ver-tebrate germline transgenesis and expression has been achieved using transposons [4]. Important considerations for the use of transposons for germline transgenesis are the carrying capacity of the transposon vector, its ability to generate multiple, independent insertion events in one embryo, the tendency of introduced genes to be expressed within such vectors, and target site choice for integration. Obviously, vectors that can tolerate large DNA inserts have the advantage of allowing large cDNAs and regula-tory sequences or multiple genes to be co-integrated at one position. As mentioned above, if multiple insertions per embryo are generated, then the total number of inde-pendent transgenesis events is increased, perhaps increas-ing the chance that one can be found which expresses at the desired level. Another important, but so far largely
unexplored issue, is the effect of transposon vector sequences on transgene expression. It remains possible that inverted terminal repeat sequences from these vectors would be recognized by host cell machinery as repetitive sequence and thus genes in cis could be silenced by meth-ylation. For this reason, and to avoid mobilization of endogenous elements, it may be best to use transposons in vertebrate species that have been isolated from very dis-tantly related species. Finally, the tendency of a given transposon system to integrate vector DNA in transcribed versus non-transcribed chromatin may affect the overall transgene expression rate and tendency to mutate endog-enous genes by insertion.
endogenous genes is critical for this application of trans-poson technology.
The third application of transposon technology is the insertional mutation of endogenous genes for genetic screens. This approach has been extensively pursued for genetic studies in invertebrates such as Drosophila mela-nogaster and in many plant species [7–10]. The advantage of such a system is that germline mutations caused by transposon insertion are molecularly tagged, greatly facil-itating their molecular identification. This advantage is in stark contrast to germline mutagenesis using X-irradiation or chemicals, which often do not leave any easily identifi-able trace of their activity or create such large chromo-somal rearrangements or deletions as to affect multiple genes at one time. One of the best chemical mutagens for vertebrate germline mutagenesis is ethyl nitrosourea (ENU), an ethylating agents that generally causes single base pair mutations [11]. When used to treat fertile male mice for instance, ENU can mutate the average locus in one in every 750 to one in every 1500 gametes, making it an incredibly powerful mutagen [11]. Even so, each dom-inant or recessive mutation must be mapped to high reso-lution using meiotic recombination until a sufficiently small critical region of the genome is identified, usually much less than 1 cM in size. At this point, candidate genes in the region are laboriously screened for mutations by directly sequencing exons and splice junctions. Thus, gene identification remains a major bottleneck in ENU muta-genesis projects. Another advantage of transposon inser-tional mutagenesis is the ability to engineer special vectors that can express a reporter molecule in a context-dependent manner. Such vectors are often called "gene-traps" and can be designed to express a reporter gene if and only if insertion occurs in the right place and orienta-tion. The three major types of gene-traps are: 1. enhancer traps, 2. promoter or 5' gene traps and 3. polyadenylation site or 3' gene traps. Enhancer traps contain a very weak promoter driving a reporter molecule, which if inserted near a gene can come under the influence of endogenous enhancer elements. The result is the temporal and/or tis-sue-specific expression of the reporter in transgenic ani-mals. Enhancer trapping has been widely used in
Drosophila melanogaster to create lines of flies expressing the GAL4 transcription factor in tissue specific patterns. In this way, a large number of so-called GAL4 driver lines can be bred with lines carrying other transgenes under the control of promoters containing upstream activating sequences (UAS) responsive to GAL4 [12]. Similar sys-tems could be devised for use on vertebrate model organ-isms, perhaps based on GAL4/VP16 or tetracycline transactivator/VP16 fusions [13]. Promoter or 5' gene-traps created using transfected plasmid DNA or retroviral transduction have been widely pursued in cultured mouse embryonic stem (ES) cells to create large libraries of
clones, each with one disrupted gene [14–17]. For this work, the vectors contain a splice acceptor followed by a gene conferring resistance to an antibiotic such as puro-mycin or G418. Thus, insertions into genes can be selected for in culture and the inserted gene is mutated since splic-ing into the plasmid or retroviral vector produces a trun-cated fusion transcript. Alternatively, 3' or polyadenylation site traps contain an internal promoter driving a reporter molecule or antibiotic resistance gene followed by a splice donor but lack any splice acceptor or polyadenylation sequence [17]. If the vector inserts into a gene-free region, then it's reporter protein will not be expressed, because the transcript is neither spliced nor polyadenylated, resulting in poor export from the nucleus and RNA instability. If the vector inserts within a gene and in the same orientation as that gene, then splicing to downstream exons results in the production of a stable fusion transcript that is spliced and polyadenylated, thus the reporter protein is expressed. In this way, ES clones can be selected that harbor insertions into genes, even if that gene is not expressed in ES cells. It should be noted that 3' traps are not thought to be highly mutagenic unless they contain an upstream 5' trap to efficiently truncate the endogenous gene [17]. Each of these three gene trapping vectors could be pursued for use in transposons for verte-brate germline mutagenesis. For this application, mobili-zation of chromosomally resident transposons in transgenic animals has the advantage of allowing new gene insertions to be obtained simply by breeding. Thus, true forward genetic screens could be performed in which phenotypes were identified first and then gene identifica-tions made second. A critical issue for using any transpo-son for the purpose of germline insertional mutagenesis is the frequency of transposon mobilization. Too few inser-tions per gamete means that too few useful offspring are obtained, thus wasting animal resources and resulting in inefficiency. Too many insertions per gamete means that multiple, perhaps linked, genes could be mutated in the same animal. This will complicate identification of the insertion responsible for a given phenotype. Also critical for this application of transposon technology is a thor-ough understanding of the target site choice of the vector within the genome, with regard to the location of the donor site, transcribed versus non-transcribed regions, and within the genes into which it becomes inserted. For instance, some transposons, when mobilized from a chro-mosomal site, tend to insert within a local region near the donor site [18,19] (Carlson et al., Genetics, In Press). This effect will bias toward mutagenesis in a particular region of the genome. While this could be an advantage, local hopping would have an influence over a whole-genome screen.
described above in vertebrate cell lines or animals. In the conclusion section, some predictions are made as to future development of these and other transposons for creating and manipulating transgenic animals.
L1 Elements
Among the vertebrate retrotransposable elements, the long interspersed repetitive elements (LINEs) have been extensively characterized and have been engineered for gene transfer and insertional mutagenesis [3]. LINEs are particularly abundant in the human genome with approx-imately 350,000 copies. Almost all of these elements are inactive due to mutations in the promoter or one or both of its two open reading frames. However, LINEs remain active in the human genome, and can in fact cause inher-ited genetic disorders [3]. It is estimated that up to 1% of human genetic mutations are due to LINE insertions. In contrast, mouse LINEs are much more active, causing per-haps 10% of mouse germline mutations [3,20]. The human L1 element promoter is active only in germline cells, but a heterologous promoter can be used to drive expression of the L1 transcript [21]. By cloning an L1 ele-ment, from an insertion into the factor VIII gene from a hemophilia A patient, Dr. Haig Kazazian's laboratory was able to isolate an active L1 element [21]. This vector was cloned and altered so that a reverse orientation, intron-interrupted, neomycin resistance gene (NEO) with a het-erologous promoter was placed in the 3' UTR. After one round of transcription, splicing, reverse transcription, and integration, the NEO gene can be expressed and confers resistance to G418 selection. This cloned and altered L1 retrotransposon vector was shown to have activity in cul-tured human and mouse cells [21]. An altered version of this L1 retrotransposon was then constructed in which NEO was replaced by the gene for the green fluorescent protein (GFP) driven by the acrosin promoter [22]. In this way, retrotranspositon could be followed by the appear-ance of GFP positive cells. When transgenic mice were created with this L1/GFP element, an examination of sperm cells revealed a fairly high frequency of GFP+ cells. When these mice were bred to wild type females, approx-imately one in sixty offspring harbored new L1 vector insertions [22]. Potential advantages of this system include the fact that the entire genome potentially could be mutagenized using the L1 vector without bias to one chromosome or chromosome region, more insertions per genome might be obtained using recently identified more active L1 elements [23], and increases in transcription might provide increases in insertion rate. Current disad-vantages include the relatively low number of insertions per gamete obtained so far, the fact that the 3' UTR of the L1 vector may tolerate only a limited amount of foreign sequence, the frequent 3' truncation of L1 vectors upon retrotransposition [21], and the fact that L1 insertion is often accompanied by deletion of sequences at the
inser-tion site [24]. L1 vectors might be useful for introducing genes into somatic cells and have been cloned into aden-oviral vectors, which can efficiently deliver L1 vectors to human cultured cell lines, resulting in retrotransposition into the genome [25].
hAT Elements: Tol2
In 1996, Dr. H. Hori's lab in Japan reported the isolation of a vertebrate transposon of the hobo-Activator-Tam3 (hAT) family of transposable elements inserted into the tryosinase gene of a strain of albino Medaka fish [26]. Sub-sequent research has shown that this element is indeed autonomous [27,28]. Tol2 is the first, and to date only, naturally occurring active cut-and-paste transposon iso-lated from vertebrates. However, an examination of endogenous Tc1/mariner-like zebrafish transposons sug-gests that some are active [29]. The Tol2 transposase gene is produced from a singly spliced transcript. The Tol2 transpoase gene can be produced from a heterologous promoter, while the transposon itself can be altered so that it contains a foreign gene cassette [27]. This two-part system can thus be used for a variety of gene transfer pur-poses. To date highly efficient germline transgenesis of the Zebrafish, Dana rario, has been achieved by co-injection of in vitro transcribed mRNA for the transposase with trans-poson vector DNA into the one cell embryo [27]. This method helps provide a system for efficient fish transgen-esis, which is normally very inefficient by injection of plasmid DNA alone (Steve Ekker, personal communica-tion). The Tol2 element may be active in mammalian cells, especially given the precedent for activity in human and mouse cells of a fish cut-and-paste transposon pro-vided by study of Sleeping Beauty [4,30,31]. Indeed, Tol2 mediated excision, but not full transposition, has already been demonstrated in human and mouse cells [32]. It remains to be determined if the Tol2 transposase can be expressed as a transgene in the germline of transgenic ani-mals and allow the mobilization of chromosomally resi-dent transposon vectors. Potential advantages of Tol2 for germline mutagenesis include the fact that hAT elements have been shown to have the capacity to transpose very large vectors [33], and the fact that it is a naturally occur-ring element and so may be more active than recon-structed elements. Recent studies of the hobo element suggest that it has distinct hot spots for integration and that the percentage of the genome available for insertion is more restricted than for other transposable elements such as the P element [33,34].
Tc1/mariner Elements: Mariner (Himar1, Mos1), Tc1, Tc3, Minos, Sleeping Beauty
[1]). These elements have been found in all vertebrate genomes examined, but are in all cases defective, contain-ing frameshift mutations, stop codons and small or large deletions in the transposase gene. A lot of research has been invested into this family of transposons based upon investigations into the active proto-typical Tc1, from
Ceanorhabditis elegans, and mariner elements, such as Mos1 from Drosophila mauritania. Members of this family contain a transposase with a DNA binding domain dis-tantly related to the paired-box DNA binding domain. In addition, a catalytic DDE domain has been identified and critical amino acids in this domain have been discovered [1].
The mariner and Tc1 transposases have been purified, and remarkably, can be combined with a transposon DNA substrate, which will then undergo transposition in vitro
without a requirement for any other proteins [35,36]. Two closely related versions of mariner have been well studied, Mos1 and Himar1, but are widespread in insects [37]. The Himar1 element is a consensus sequence based on several clones isolated from the horn fly, Haematobia irritans, and Mos1 was isolated from Drosophila mauritania [35]. The wide distribution of Tc1/mariner transposons, the ability of mariner to function in vitro in purified form and in dis-tantly related species, suggested that such elements might require no co-factors for transposition in cells, or use very highly conserved host factors. For example, Mos1 has been introduced into the distantly related insect species, such as the yellow fever mosquito Aedes aegypti [38]. For these reasons, great enthusiasm exists for adapting Tc1/ mariner-like transposons for use in vertebrate species. Indeed mariner, Tc1 and Tc3 show activity in cultured mouse and human cell lines [39,40]. A screen for hyper-active mutants in E. coli resulted in the identification of amino acid substitutions that improve the activity of Himar1 in cultured cells [41]. The Mos1 mariner transpo-son has been used to achieve germline transgenesis in the chicken [5]. Both mariner and Tc3 has been used to achieve germline transgenesis in zebrafish [42,43]. The purified Himar1 transposase efficiently catalyzes inter-plasmid transposition [35]. The same kind of interplas-mid transposition assay was used in a human 293T embryonic kidney cell line, showing that Himar1 transpo-sition can occur in mammalian cells [44]. While Himar1 is primarily used for insertional mutagenesis in bacteria and mycobacteria, its potential for vertebrate gene transfer and insertional mutagenesis remains unclear. It remains to be determined if the active mariner elements, Tc1, or Tc3 can be used to mobilize chromosomally resident transposon vectors in vertebrates.
The Minos element, a Tc1/mariner-like transposon from
Drosophila hydei, has been shown to be active in cultured human cell lines [45,46]. A gene-trap Minos transposon
has been constructed and a library of insertions in HeLa cells were obtained [46]. The Minos transpoase gene has been expressed in transgenic mice from B cell lineage spe-cific and male sperm cell spespe-cific promoters [47,48]. When these mice were crossed with transgenic mice carry-ing Minos transposon vectors, transposition occurred in B cells and in the male germline respectively. Germline insertions occurred in rough one in every ten offspring. Several germline insertions were cloned and each was found to be on a different chromosome [48]. These data suggest that Minos might be useful for mouse germline mutagenesis and that no distinct preference for local transposition occurs using this transposon system. Thus, genome-wide insertional mutagenesis with Minos is a real possibility in the mouse and possibly other species as well.
experi-ments and transposon transgenes could be expressed. However, the transgenes within transposon vectors seem to be subject to position effect variagation, just as are standard mouse transgenes. These data showed that mam-malian germline transgenesis by transposition is possible and suggest that the SB system might find utility in other mammalian species in which transgenesis is difficult. Another application of SB has been somatic cell transgen-esis. SB can be used for stable, long-term gene transfer and expression into the liver of adult mice [51]. In some exper-iments, the SB transposon vector and transposase trans-gene have been delivered by so-called "hydrodynamic therapy". In this approach, 10% of the weight of the mouse of DNA-containing Ringer's solution is injected via the tail vein into mice in less than ten seconds. For the average mouse this is roughly 2–2.5 milliliters. The result-ant transient increase in venous pressure within the liver is thought to result in extravasation of the plasmid DNA, which is efficiently taken up by ~25% of hepatocytes [52]. The Factor IX gene was cloned into an SB transposon vec-tor and delivered to hemophilia B knockout mice using this technique by Dr. Mark Kay's laboratory [51]. Long-term expression required co-delivery of the Factor IX transposon and an active SB10 transposase gene on another plasmid. Similar success has been achieved for long-term gene transfer of the FAH gene into knockout mouse liver [53], and the LAMB3 gene into cultured human skin cells from patients with junctional epidermo-lysis bullosa syndrome to correct this disorder in xenografted nude mice [54]. Dr. Mark Kay's group also has developed "binary" gene therapy vectors in which both the SB transposon vector and SB10 transposase gene were delivered to hepatocytes using adenoviral vectors, which by themselves only result in transient gene transfer and expression [31]. Interestingly, efficient gene transpo-sition required recombinase-mediated excision and circu-larization of the transposon vector from the linear adenoviral DNA, suggesting that circularized transposon vector DNA is more efficiently transposed, at least in this environment [31]. Dr. Scott McIvor's laboratory has developed methods for long term gene transfer and expression into adult mouse lung using SB vector and SB10 transposase DNA complexed with polyethyleneimine (PEI), a polycationic, branched mole-cule (Beleur et al., Molecular Therapy, In Press). Finally, in the area of germline insertional mutagenesis, we have demonstrated that SB transposons present in chromo-somes of SB transposase transgenic mice will efficiently undergo transposition in germline cells, such that off-spring from these mice are obtained with new transposon insertions [55]. Similar results were published in 2001 by two other labs using SB, one in Japan and one in the Neth-erlands [19,56]. Three papers all report essentially similar results, with differences in the average number of new transposon insertions obtained. The ubiquitously
expect to achieve a 1X coverage of a 10 Mbp region of the genome (with one insertion every 10 kb) in as few as 1000 mice. We have begun to characterize two embryonic lethal mutations caused by endogenous splicing disruption in mice carrying intron-inserted SB gene-trap transposons. It is clear from these analyses that SB and probably other cut-and-paste transposons have potential utility as ran-dom germline mutagens for forward genetic screens.
Conclusions and Future Directions
It is very likely that transposon technology will have an impact in one or more of the three applications described in this review over the next several years. This progress may involve use of one or more of the transposon systems described above or may involve new transposon systems. The precedent set by work on SB shows that evolutionarily defunct transposons can be "resurrected" using reverse evolutionary principals. This means that many other potentially useful transposons could be derived from a purely informatics based approach using sequenced genomes. A rich source of new transposon systems could be generated in this way, some of which may have attributes more suited to one or another application. Alternately, it is likely that many species contain active elements, such as Tol2 and L1, since we see the evidence of their past activity in all genomes examined. Again, the search for these active elements will be a useful outcome of ongoing genome projects. Mammalian or avian germ-line transgenesis by transposition could have an impact on several agriculturally relevant species. Transgenesis for most of these species is currently very difficult or impossi-ble. Given the technical challenges of harvesting, manipu-lating, and injecting early embryos from some of these species, it is worth considering sperm transgenesis by transposition. This process has been achieved in mice via micro-injection [57], and might be made more efficient using transposon vectors. While the risk of insertional mutagenesis, specifically activation of endogenous proto-oncogenes and cancer, are present with transposons used in gene therapy [58], it remains to be determined if they are low of enough compared to the benefits that transpo-son-based gene therapy may bring. However, it is clear from work using SB that the concept is sound from a technical standpoint. Finally, transposon-mediated inser-tional mutagenesis of the mouse germline is clearly possible with SB, Minos and probably Tol2. Transposon-based germline mutagenesis might also be considered for other species, particularly those for which ES cells cannot be easily obtained. In the mouse, however, the best use of each system probably depends upon the type of screen desired. The maximal number of insertions per gamete that can be reliably obtained must be determined. The ideal sequences for gene-trapping must be identified. Applications such as local saturation mutagenesis or chro-mosome engineering by placement of LoxP sites within
transposon vectors are certainly possible. It is hoped that such approaches can led to significant contributions to functional annotation of the mammalian genome in the future.
Acknowledgments
The author thanks Drs. Perry Hackett, Steve Ekker, Scott McIvor, and other members of the University of Minnesota Arnold and Mabel Beckman Center for Transposon Research for helpful suggestions and technical review. This work was partially supported by The Arnold and Mabel Beck-man Foundation, the University of Minnesota Academic Health Center and Graduate School, and NIH grant R01 DA14764.
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